In vitro Pharmacodynamics of Antifungal Agents in the Treatment of

84 Pages·2010·2.03 MB·English

by  Anders Lignell

List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.

I Lignell, A., Johansson, A., Löwdin, E., Cars, O., Sjölin, J.
(2007) A new in vitro kinetic model to study the
pharmacodynamics of antifungal agents: inhibition of the
fungicidal activity of amphotericin B against Candida albicans
by voriconazole.
Clinical Microbiology and Infection 13: 613-619

II Lignell, A., Löwdin, E., Cars, O., Sjölin, J. (2008)
Characterization of the inhibitory effect of voriconazole on the
fungicidal activity of amphotericin B against Candida albicans
in an in vitro kinetic model.
Journal of Antimicrobial Chemotherapy 62: 142-148

III Lignell, A., Löwdin, E., Cars, O., Sanglard, D., Sjölin, J. (2010)
Voriconazole-induced inhibition of the fungicidal activity of
amphotericin B in Candida strains with reduced susceptibility
to voriconazole: an effect not predicted by the MIC value alone.
Submitted

IV Lignell, A., Löwdin, E., Cars, O., Chryssanthou, E., Sjölin, J.
(2010) Posaconazole in human serum: A greater
pharmacodynamic effect than predicted by the non-protein-
bound serum concentration.
Submitted
Reprints were made with permission from the respective publishers.

Contents
Introduction.....................................................................................................9
Pathogen.....................................................................................................9
Epidemiology of invasive Candida infections...........................................9
Risk factors for invasive Candida infections...........................................10
Clinical symptoms of invasive Candida infections..................................10
Diagnosis of invasive Candida infections................................................11
Treatment of invasive Candida infections...............................................12
Mechanisms of action of antifungal agents..............................................14
Triazoles..............................................................................................14
Polyenes...............................................................................................15
Echinocandins......................................................................................15
Antimetabolites....................................................................................15
Antifungal pharmacodynamics.................................................................15
Time course of antimicrobial activity..................................................16
Pharmacodynamic parameters predictive of efficacy..........................18
Pharmacodynamic target.....................................................................19
The clinical relevance of pharmacodynamics......................................19
The role of protein binding..................................................................20
Combination treatment of invasive Candida infections...........................21
In vitro studies.....................................................................................22
Animal model studies..........................................................................22
Clinical trials........................................................................................24
Aims of the studies........................................................................................25
Materials and methods..................................................................................26
Fungal strains...........................................................................................26
MIC determination...................................................................................26
Characterisation of mechanisms of resistance of C. albicans strains
(Study III).................................................................................................28
Medium....................................................................................................28
Antifungal agents.....................................................................................28
In vitro kinetic model...............................................................................28
Pharmacokinetic analysis (Studies I and II).............................................29
Protein binding and concentration analysis of posaconazole in human
serum (Study IV)......................................................................................31
Pharmacodynamic experiments................................................................32
Study I..................................................................................................32
Study II................................................................................................33
Study III...............................................................................................34
Study IV...............................................................................................35
Antifungal carryover................................................................................36
Statistics and calculations.........................................................................37
Results...........................................................................................................38
MIC determination...................................................................................38
Characterisation of mechanisms of resistance in strain CA4 (Study
MIC256
III)............................................................................................................40
Pharmacokinetic analysis (Studies I and II).............................................40
Protein binding and concentration analysis of posaconazole in human
serum (Study IV)......................................................................................42
Pharmacodynamic experiments................................................................42
Study I..................................................................................................42
Study II................................................................................................43
Study III...............................................................................................48
Study IV...............................................................................................53
Discussion.....................................................................................................56
Testing antifungal combinations..............................................................56
In vitro kinetic model...............................................................................56
In vitro pharmacodynamics of voriconazole and amphotericin B against
Candida spp..............................................................................................57
Clinical relevance of the voriconazole-amphotericin B interaction.........60
In vitro serum pharmacodynamics of posaconazole against Candida spp.
..................................................................................................................63
Conclusions...................................................................................................66
Future perspectives.......................................................................................67
Summary in Swedish – Farmakodynamiska studier av svampläkemedel mot
Candida-infektioner......................................................................................68
Acknowledgements.......................................................................................70
References.....................................................................................................72
Abbreviations
AMB amphotericin B
ANOVA analysis of variance
AUC area under the concentration curve
BSI bloodstream infection
CCUG Culture Collection University of Göteborg
CFU colony forming unit
CLSI Clinical and Laboratory Standards Institute
C concentration maximum
max
C concentration minimum
min
EUCAST European Committee on Antimicrobial
Susceptibility Testing
FLU fluconazole
HPLC high-performance liquid chromatography
IDSA Infectious Diseases Society of America
LC-MS liquid chromatography-mass spectrometry
MFC minimum fungicidal concentration
MIC minimum inhibitory concentration
PAFE post-antifungal effect
PBS phosphate-buffered saline
PCR polymerase chain reaction
POS posaconazole
RNA ribonucleic acid
RPMI Roswell Park Memorial Institute medium
SD standard deviation
SE standard error
t half-life
1/2
VRC voriconazole

Introduction
Candida species can cause a variety of infections in humans from superficial
mucosal or cutaneous disease to life-threatening invasive infection with
hematogenous dissemination to practically any organ. This thesis focuses on
invasive Candida infections. Nevertheless, several aspects of the treatment
of invasive Candida infections can be applied to superficial disease.
Pathogen
Candida species are eukaryotic organisms belonging to the kingdom Fungi.
Fungi are distinguished from other eukaryotes by a rigid cell wall composed
of chitin and glucan and a cell membrane in which ergosterol is substituted
for cholesterol as the major sterol component.1 Candida species are yeastlike
organisms, i.e. they exist mainly in unicellular form. They are small (4-6
μ m), oval-form cells that reproduce by budding. The majority form smooth,
creamy white colonies on agar plates.2 Moulds, unlike the yeasts, are
multicellular fungi that form tubular structures called hyphae that grow by
branching or apical extension and form fuzzy, hairy colonies on agar.1, 2
Epidemiology of invasive Candida infections
Candida spp. are found in humans, animals and in nature. The organisms are
known colonisers of humans and mainly found in the gastrointestinal tract
from the mouth to the rectum and may be found in the vagina, in the urethra,
on the skin and under the nails.1
The majority of Candida infections are endogenous, i.e. the source of
infection is the normal flora of the patient that takes the advantage of the
opportunity to cause infection.1, 2 Exogenous transmission of Candida spp.
from health care workers to patients, from patient to patient or from the
hospital environment to the patient has also been described.3-6
There are at least 17 species of Candida reported to cause human
disease.7, 8 Among the various species of Candida, C. albicans, C. glabrata,
C. parapsilosis, C. tropicalis and C. krusei cause over 90% of all invasive
Candida infections with C. albicans being the most common pathogen.8, 9 C.
glabrata (in adults) and C. parapsilosis (in children) are the second most
9
commonly isolated species.8 In Sweden, C. albicans also predominates and
causes approximately 60-70% of invasive candidiasis, C. glabrata causes
15-20% and the rest are caused by other non-albicans species.10, 11
During the past decades, there has been an increase in the number of
invasive Candida infections, which is largely due to the increasing size of
the population at risk of contracting candidiasis.12, 13 Data that are more
recent indicate that the increase may have stopped but there seems to be an
increase in the proportion of fluconazole-resistant non-albicans strains
causing invasive Candida infections.12, 14 Invasive Candida infection is the
fourth most common cause of nosocomial bloodstream infections (BSI) in
the USA, accounting for 8-10% of all BSIs acquired in hospital.15 In a
number of studies the incidence rates for invasive Candida infections vary
from 2 to 20 per 100,000 population.8, 12 Although there is treatment
available, the crude (all-cause) mortality associated with severe Candida
infections is substantial.8, 16 The attributable mortality rate, i.e. the mortality
traceable directly to candidiasis and not to underlying disease is difficult to
study but ranges in various investigations from 15-50% in adult patients.17-21
Risk factors for invasive Candida infections
Risk factors for invasive Candida infections are immunosuppressive
diseases or treatment, factors promoting Candida colonisation and factors
providing an access route to the vascular system for the pathogen.
Independent risk factors are exposure to broad-spectrum antimicrobial
agents, cancer chemotherapy, mucosal colonisation by Candida spp.,
vascular catheters, total parenteral nutrition, neutropenia, prior surgery
(especially gastrointestinal surgery), gastric acid suppression and renal
failure or haemodialysis.8, 12 Further, invasive Candida infections are more
common in children under one year of age and in patients over 65 years.8, 12
With the combination of risk factors, the probability of infection increases
exponentially.22
Clinical symptoms of invasive Candida infections
Invasive Candida infections originate from a site of colonisation from where
the pathogen invades the bloodstream and causes candidemia. At this time,
the patient presents symptoms of sepsis, which in some cases progress to
severe sepsis or septic shock with hypotension, organ dysfunction and
hypoperfusion. More often, especially in non-neutropenic intensive care unit
patients, a moderate inflammatory reaction takes place causing fever and a
rise in inflammatory parameters as well as progressive organ dysfunction.
There is, in fact, no characteristic constellation of signs and symptoms to
10